The overall objectives of this program are to obtain information about: (1) Transport of folate compounds (including the cancer chemotherapeutic agent, Methotrexate) into L1210 and Lactobacillus casei cells; and the (2) Mechanism of folate-dependent enzymes, including dihyrofolate reductase, and methionine synthetase. Projects to be undertaken under (1) include: (a) purification and characterization of the 5-methyltetrahydrofolate/MTX binding protein from L1210 cell membranes; (b) examination of the relationship between folate and adenine transport in L1210 cells; and (c) studies on the folate-binding protein from L. casei including its structure, behavior in Triton micelles, reconstitution in liposomes, energy-coupling, and high and low affinity forms. Under (2), the L1210 dihydrofolate reductase will be characterized with respect to (a) the nature and spatial relationship of the substrate binding sites; and (b) the MTX-binding properties of naturally-occurring and synthetic multiple forms. Methioine synthetase will be purified, via affinity chromatography from bovine liver and L1210 cells, and the role of this enzyme (along with vitamin B12 and transcobalamin-II) in the replication of L1210 cells will be examined. The methionine synthetase system from Escherichia coli K-12 (B-12-protein and two flavoproteins) will be further characterized with respect to: (a) sulfhydryl groups on the B12-protein; (b) reduction potential of the bound B12; (c) mechanism of electron transfer; and (d) physical interaction of the three proteins.